Bottom Line:
Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region.Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae.The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system.

Affiliation: BioMediTech, University of Tampere, Tampere, Finland; School of Medicine, University of Tampere, Tampere, Finland.

ABSTRACTCarbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.

pone.0134263.g001: Bayesian phylogenetic tree of CARP sequences.Numbers at the nodes indicate posterior probabilities, and branch lengths are proportional to distances. A table of full details of the sequences is given as S1 Table.

Mentions:
Zebrafish and other ray-finned fishes have two CA10 orthologs, ca10a and ca10b, as reported earlier [6]. ca10a is highly similar to mammalian CA10 (90% identity to human CARP X at protein level), whereas ca10b is slightly more diverged, yet highly similar (75% identity between human CARP X and zebrafish CARP Xb) (Table 5). In addition, the phylogenetic branching pattern (Fig 1) and the appearance of the alignment (Fig 2) clearly support the notion that fish CARP Xa and Xb are closer to tetrapod CARP X than to CARP XI. The position of the two CARPs of lamprey (Lethenteron) indicates that they have duplicated from a single ancestor in the cyclostome lineage, and that the duplication to CARP X and CARP XI took place only in the jawed vertebrates.

pone.0134263.g001: Bayesian phylogenetic tree of CARP sequences.Numbers at the nodes indicate posterior probabilities, and branch lengths are proportional to distances. A table of full details of the sequences is given as S1 Table.

Mentions:
Zebrafish and other ray-finned fishes have two CA10 orthologs, ca10a and ca10b, as reported earlier [6]. ca10a is highly similar to mammalian CA10 (90% identity to human CARP X at protein level), whereas ca10b is slightly more diverged, yet highly similar (75% identity between human CARP X and zebrafish CARP Xb) (Table 5). In addition, the phylogenetic branching pattern (Fig 1) and the appearance of the alignment (Fig 2) clearly support the notion that fish CARP Xa and Xb are closer to tetrapod CARP X than to CARP XI. The position of the two CARPs of lamprey (Lethenteron) indicates that they have duplicated from a single ancestor in the cyclostome lineage, and that the duplication to CARP X and CARP XI took place only in the jawed vertebrates.

Bottom Line:
Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region.Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae.The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system.

Affiliation:
BioMediTech, University of Tampere, Tampere, Finland; School of Medicine, University of Tampere, Tampere, Finland.

ABSTRACTCarbonic anhydrase related proteins (CARPs) X and XI are highly conserved across species and are predominantly expressed in neural tissues. The biological role of these proteins is still an enigma. Ray-finned fish have lost the CA11 gene, but instead possess two co-orthologs of CA10. We analyzed the expression pattern of zebrafish ca10a and ca10b genes during embryonic development and in different adult tissues, and studied 61 CARP X/XI-like sequences to evaluate their phylogenetic relationship. Sequence analysis of zebrafish ca10a and ca10b reveals strongly predicted signal peptides, N-glycosylation sites, and a potential disulfide, all of which are conserved, suggesting that all of CARP X and XI are secretory proteins and potentially dimeric. RT-qPCR showed that zebrafish ca10a and ca10b genes are expressed in the brain and several other tissues throughout the development of zebrafish. Antisense morpholino mediated knockdown of ca10a and ca10b showed developmental delay with a high rate of mortality in larvae. Zebrafish morphants showed curved body, pericardial edema, and abnormalities in the head and eye, and there was increased apoptotic cell death in the brain region. Swim pattern showed abnormal movement in morphant zebrafish larvae compared to the wild type larvae. The developmental phenotypes of the ca10a and ca10b morphants were confirmed by inactivating these genes with the CRISPR/Cas9 system. In conclusion, we introduce a novel zebrafish model to investigate the mechanisms of CARP Xa and CARP Xb functions. Our data indicate that CARP Xa and CARP Xb have important roles in zebrafish development and suppression of ca10a and ca10b expression in zebrafish larvae leads to a movement disorder.